Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in ﻿the Netherlands

Abstract Diaporthe (Diaporthaceae, Diaporthales) is a common fungal genus inhabiting plant tissues as endophytes, pathogens and saprobes. Some species are reported from tree branches associated with canker diseases. In the present study, Diaporthe samples were collected from Alnusglutinosa, Fraxinusexcelsior and Quercusrobur in Utrecht, the Netherlands. They were identified to species based on a polyphasic approach including morphology, pure culture characters, and phylogenetic analyses of a combined matrix of partial ITS, cal, his3, tef1 and tub2 gene regions. As a result, four species (viz. ﻿﻿Diaporthepseudoalnea sp. nov. from Alnusglutinosa, Diaporthesilvicola sp. nov. from Fraxinusexcelsior, D.foeniculacea and D.rudis from Quercusrobur) were revealed from tree branches in the Netherlands. Diaporthepseudoalnea differs from D.eres (syn. D.alnea) by its longer conidiophores. Diaporthesilvicola is distinguished from D.fraxinicola and D.fraxini-angustifoliae by larger alpha conidia.

Species of Diaporthe are known to cause plant diseases including dieback, canker, leaf spot, fruit rot, pod blights and seed decay. For example, D. citri, D. cytosporella and D. foeniculina caused melanose and stem end rot diseases of Citrus spp. (Udayanga et al. 2014), while Daporthe lithocarpi caused leaf spot disease of Castanea henryi in China . Up to 19 Diaporthe species were confirmed to be associated with pear cankers in China (Guo et al. 2020), and eight species of Diaporthe were found to be the casual agents of Chinese grapevine dieback (Manawasinghe et al. 2019). Seven Diaporthe species were reported from blueberry twig blight and dieback diseases in Portugal (Hilário et al. 2020). Diaporthe biconispora and an additional six species were identified as endophytes from healthy Citrus tissues in China (Huang et al. 2015). Diaporthe constrictospora and an additional 11 species were isolated as saprobes from dead wood in karst formations in China (Dissanayake et al. 2020).
Diaporthe species were previously classified mainly based on host association and morphology (Rehner and Uecker 1994;Santos and Phillips 2009;Udayanga et al. 2011Udayanga et al. , 2014. However, several taxonomic studies of Diaporthales proved that phylogeny based on multiple genes is suitable to separate species (Voglmayr et al. 2012(Voglmayr et al. , 2017Fan et al. 2018;Jiang et al. 2019Jiang et al. , 2020Voglmayr 2019, 2020). Species of Diaporthe are now characterised and circumscribed both by morphology and phylogeny of multi-locus DNA data, which revealed many cryptic species in recent years (Diogo et al. 2010;Lombard et al. 2014;Gao et al. 2016Gao et al. , 2017Long et al. 2019;Yang et al. 2020Yang et al. , 2021Zapata et al. 2020;Huang et al. 2021). To clarify the species boundaries of the Diaporthe eres complex, the Genealogical Phylogenetic Species Recognition principle (GCPSR) and the coalescent-based model Poisson Tree Processes (PTPs) were employed, which suggested that the Diaporthe eres species complex actually represents only a single species, D. eres (Hilário et al. 2021).
In the present study, Diaporthe samples from cankered branches of several tree species were collected in the Netherlands, and identified based on modern taxonomic approaches. As a result, two new species and two known species were identified, and the new species are described and illustrated herein.

Collection, examination and isolation
The fresh specimens of cankered branches were sampled from Alnus glutinosa, Fraxinus excelsior and Quercus robur in Utrecht, the Netherlands. Morphological characteristics of the conidiomata were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 conidiomata were sectioned, and 50 conidia were randomly selected for measurement using a Leica compound microscope (LM, DM 2500). Isolates were obtained by removing a mucoid conidial mass from conidiomata, spreading the suspension onto the surface of 1.8 % potato dextrose agar (PDA), and incubated at 25 °C for up to 24 h. Single germinating conidia were removed and plated onto fresh PDA plates. Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and conidiomata structures. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://www. cfcc-caf.org.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

DNA extraction, PCR amplification and phylogenetic analyses
Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). DNA was checked by electrophoresis in 1 % agarose gel, and the quality and quantity were measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Five partial loci, including the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the calmodulin (cal), the histone H3 (his3), the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) genes were amplified by the primer pairs and polymerase chain reaction (PCR) process listed in Table 1. The PCR products were assayed via electrophoresis in 2 % agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a Big- The quality of the amplified nucleotide sequences was checked and the sequences assembled using SeqMan v.7.1.0. Reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Diaporthe (Dissanayake et al. 2021;Gao et al. 2021;Huang et al. 2021;Sun et al. 2021, Wang et al. 2021Yang et al. 2021). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using MEGA 7.0.21. The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.
The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014), employing a GTRGAMMA substitution model with 1000 bootstrap replicates. While BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1000000 generations and trees, were sampled every 100th generation, resulting in a total of 10000 trees. The first 25 % of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank and are listed in Table 2.

Phylogenetic analyses
The five-gene sequence dataset (ITS, cal, his3, tef1 and tub2) was analysed to infer the interspecific relationships within Diaporthe. The dataset consisted of 307 sequences including one outgroup taxon, Diaporthella corylina (CBS 121124). A total of 2649 characters including gaps (516 for ITS, 576 for cal, 526 for his3, 507 for tef1 and 524 for tub2) were included in the phylogenetic analysis. Of these characters, 844 were constant, 318 were variable and parsimony-uninformative, and 1487 were parsimony-informative. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). Isolates from the present study formed four individual clades representing four species of Diaporthe, of which isolates CFCC 54192, M35, M40-1 and M84 from Quercus robur represent D. foeniculacea, while CFCC 54193 and M86 from Q. robur represent D. rudis. CFCC 54191 and M79 from Fraxinus excelsior and CFCC 54190 and M2A from Alnus glutinosa represent two new species which are here described as D. silvicola and D. pseudoalnea, respectively.        Etymology. With reference to D. alnea, which was described from the same host genus, Alnus.
Notes. Diaporthe nivosa and D. alnea were recorded from the host genus Alnus. Udayanga et al. (2014) investigated the lectotype of Diaporthe nivosa and revealed it as a Melanconis species based on a well-developed ectostromata and the ascospores characteristics, and Jaklitsch and Voglmayr (2020) treated it as a synonym of Melanconis marginalis ssp. marginalis. D. alnea has been reported from the Czech Republic, Germany, the Netherlands and the USA, and both sexual and asexual morphs have been described (Udayanga et al. 2014). However, applying the GCPSR principle, D. alnea has recently been considered to be a synonym of Diaporthe eres (Hilário et al. 2021), which has also been confirmed in our analyses where the ex-epitype isolate CBS 146.46 of D. alnea is placed within the D. eres clade (Fig. 1). Diaporthe pseudoalnea morphologically differs from D. alnea (now D. eres) by its longer conidiophores (22-68.5 × 1.5-3 μm in D. pseudoalnea vs. 9-16 × 1-2 μm in D. alnea; Udayanga et al. 2014). In our multi-gene analyses, D. pseudoalnea forms a distinct phylogenetic lineage which is placed remotely from the isolate CBS 146.46 of D. alnea (Fig. 1).
Culture characters. Colonies are initially white, aerial mycelium turning grey at edges of plate, yellowish pigmentation developing in centre, conidiomata not produced until 2 weeks.

Discussion
In this study, branch-inhabiting Diaporthe species were sampled from Alnus glutinosa, Fraxinus excelsior and Quercus robur in Utrecht, the Netherlands. Ten Diaporthe isolates were obtained and identified based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters from the natural substrates. The phylogenetic and morphological analyses revealed Diaporthe pseudoalnea sp. nov. from Alnus glutinosa, Diaporthe silvicola sp. nov. from Fraxinus excelsior, and D. foeniculacea and D. rudis from Quercus robur.
Phylogenetic analyses were conducted based on a combined DNA sequence matrix of five loci (ITS, cal, his3, tef1 and tub2) reported as useful markers to distinguish species of Diaporthe (Udayanga et al. 2014Guarnaccia et al. 2017Guarnaccia et al. , 2018aGuarnaccia et al. , 2018bTibpromma et al. 2018;Yang et al. 2020;Dissanayake et al. 2020;Huang et al. 2021;Sun et al. 2021, Wang et al. 2021). The two novel species in this study can be distinguished from the other known species by all genes studied, but most effectively by cal, his3, tef1 and tub2. The multi-locus phylogenetic analysis grouped the isolates in two new clades, which support the introduction of the new species.
The utility of host association for Diaporthe species identification is limited because several species have wide host ranges (e.g., D. ere inhabits 282 different hosts; D. rudis inhabits 44 different hosts), and multiple Diaporthe species can infect a single host (e.g., nineteen Diaporthe species are associated with pear cankers in China) (Guo et al. 2020;Farr and Rossman 2021). Thus, a polyphasic approach of morphological, cultural, ecological and molecular data to identify Diaporthe samples or to introduce new species is essential.